Prostate cancer (PCa) is the second leading cause of cancer death among American men. The high mortality is mainly caused by castration-resistant PCa (CRPC) relapsed after androgen deprivation therapy. Complete or partial loss of the PTEN tumor suppressor frequently occurs in localized and metastatic, castration-resistant PCa, suggesting that PTEN deficiency plays an important role in both PCa development and progression. Mouse genetic studies show that loss-of-function of Pten is sufficient for emergence of CRPC. However, how PTEN loss promotes development of CRPC is largely unclear. Runt-related transcription factor 2 (RUNX2) has been implicated in human PCa. However, whether or not it plays a causal role in prostate tumorigenesis and castration-resistant progression remains elusive. Motivated by our previous finding that RUNX2 overexpression correlates with decreased PTEN expression in human PCa specimens, we generate a Runx2 conditional transgenic mouse model and demonstrate that overexpression of Runx2 alone induces prostate epithelial cell transformation and low-grade prostate intraepithelial neoplasia (PIN), but in combination with Pten heterozygous deletion, causes high-grade PIN and cancer. We also show that the Akt kinase is hyperphosphorylated/hyperactivated in Runx2-Pten heterozygous tumors in mice and that concomitant RUNX2 overexpression and PTEN loss induce upregulation of the chemokine receptor CXCR7. We further demonstrate that testosterone is synthesized in murine Pten-knockout prostate tumors and PTEN-null, RUNX2-overexpressing human PCa cells and that this effect is abrogated by knockdown of RUNX2 or restored expression of PTEN in cultured PCa cells. These preliminary data lead to our central hypothesis that PTEN loss and RUNX2 overexpression work cooperatively to confer an aberrantly-activated transcriptional program of RUNX2, which in turn contributes AKT hyper activation, intratumoral androgen biosynthesis, prostate tumorigenesis and castration-resistant progression. To test this hypothesis, we will determine the molecular mechanism and downstream effectors that mediate concomitant RUNX2 overexpression and PTEN deficiency- induced prostate oncogenesis (Aim 1) and determine the cooperativity of RUNX2 overexpression and PTEN deficiency in development of aggressive and castration-resistant PCa in mice and human patient samples (Aim 2). Findings from this innovative proposal will provide a major advance in our understanding of the molecular mechanisms underlying prostate tumorigenesis and castration-resistant progression and novel aspects of PTEN tumor suppressor activity, in addition to conceptually advancing the field of RUNX2 biology in steroidogenesis and cancer, particularly in PCa. This knowledge will lay the foundation for development of new therapeutic strategies that will improve the clinical outcome of PCa patients, especially those with PTEN and RUNX2 alterations.